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ARS VETERINARIA, Jaboticabal, SP, Vol. 23, nº2, 108-115, 2007.
ISSN 0102-6380
DETECÇÃO DE S100-β E REAÇÃO DE ASTRÓCITOS
DURANTE ENCEFALITE EXPERIMENTAL
PELO VíRUS DA ESTOMATITE VESICULAR.
(S100-β DETECTION AND ASTROCYTE REACTION DURING VESICULAR STOMATITIS VIRUS
EXPERIMENTAL ENCEPHALITIS)
(DETECCIÓN DE S100-β Y REACCIÓN DE ASTROCITOS DURANTE ENCEFALITIS EXPERIMENTAL
POR EL VIRUS DE LA ESTOMATITIS VESICULAR)
G. F. MACHADO1*, P. C MAIORKA2, A. A. PINTO3, C. G. CANDIOTO4,
L. M. U. IEIRI4, A. C. ALESSI3
RESUMO
Camundongos foram sacrificados nos dias 2 (Grupo1), 6 (Grupo 2) e 20 (Grupo 3) após a inoculação do vírus (pi)
da estomatite vesicular e a proteína glial fibrilar ácida (GFAP), vimentina e S100β foram detectadas no encéfalo através de
imunoistoquímica. Os sintomas e lesões mais severos de encefalite foram observados no dia 6. Houve aumento da marcação
para GFAP em astrócitos caracterizando astrogliose. A detecção de GFAP e vimentina foram bastante evidentes no dia 6
pi, quando a marcação para vimentina foi mais intensa próxima às áreas de necrose. A proteína S100β foi intensamente
detectada no dia 6 pi em neurônios e na micróglia. Observou-se redução da marcação da GFAP em áreas de necrose. Durante
a encefalite experimental pelo vírus da estomatite vesicular e, especialmente em camundongos com sintomas sacrificados
no dia 6 pi, além de intensa marcação para GFAP e vimentina, detectamos marcação para a proteína S100β em neurônios
e na micróglia, mas não em astrócitos onde a S100β é considerada um marcador específico.
PALAVRAS-CHAVE: S100β. GFAP. Vimentina. Astrócitos. Vírus da Estomatite Vesicular.
SUMMARY
Mice were sacrificed at 2 (Group 1), 6 (Group 2) and 20 (Group 3) days post-virus inoculation (pi) and glial fibrillary
acidic protein (GFAP), vimentin and S100β staining were studied by immunohistochemistry. Symptoms and severe lesions
were observed at day six. There was an increase of GFAP staining in astrocytes characterizing astrogliosis. Detection of
GFAP and vimentin was evident after six days post-inoculation and vimentin staining was more intense around injured
areas. The S100-β protein was strongly detected at day 6-pi in neurons and microglia. Reduction in GFAP was observed
in areas of encephalic necrosis. During VSV encephalitis in the mouse model and especially in those mice with symptoms
at day six post-inoculation we showed that besides astrocytes response to VSV infection characterized by upregulation
of GFAP and vimentin, we also detected production of the S100β in neurons and microglia, but not in astrocytes, where
1 Médica Veterinária. Professora Assistente Doutora - Departamento de Clínica, Cirurgia e Reprodução Animal. FOA/Unesp - Rua
Clóvis Pestana, 793.- Araçatuba-SP.- CEP.16050-680. - E-mail.: [email protected]
2 Professor Assistente Doutor. Departamento de Patologia. Faculdade de Medicina Veterinária e Zootecnia - USP – São Paulo - SP.
3 Professor Titular - Departamento de Patologia e Parasitologia Veterinária - Faculdade de Ciências Agrárias e Veterinárias - Unesp Jaboticabal - SP.
4 Aluno de graduação do Curso de Medicina Veterinária - FO - Araçatuba - SP.
108
S100β is considered a specific marker.
KEYWORDS: Astrocytes. vesicular stomatitis vírus. GFAP. Vimentin. S100-β.
RESUMEN
Los ratones fueron sacrificados en los días 2 (Grupo 1), 6 (Grupo 2) y 20 (Grupo 3) después de la inoculación del
virus (pi) de la estomatitis vesicular. La proteína glial fibrillar ácida (GFAP), la vimentina y la S100b fueron estudiadas
por inmunoistoquímica. Los síntomas y lesiones más graves fueron observados en el día 6. Los resultados demostraron
aumento inicial de GFAP en astrocitos, seguido por la reducción en aquellos con alteraciones morfológicas degenerativas
en algunas áreas del encéfalo que correspondían a necrosis. Hubo elevación de la marcación de GFAP caracterizando
astrogliosis. La detección de GFAP y vimentina fue evidente en el día 6 pi, cuando la marcación para vimentina fue más
intensa al rededor de las áreas de necrosis. Se observó reducción en la marcación de la GFAP en las áreas de necrosis.
La S100b fue intensamente detectada en el día 6 en las neuronas y en la microglia. Durante la infección por el virus de
la estomatitis vesicular en ratones con síntomas y sacrificados en el día 6 pi, además de intensa marcación para GFAP y
vimentina en astrocitos, detectamos la proteína S100b en neuronas y en la microglia, pero no en astrocitos donde la S100b
es considerada un marcador específico.
PALABRAS-CLAVE: S-100-β. GFAP. Vimentina. Astrocitos. Virus de la Estomatitis Vesicular.
Introduction
Astrocytes are the most numerous cells within
the central nervous system. Early during development they
act as guiding structures for neurons; later they become the
main source for nutrients and growth factors in the brain,
and they are also communication partners of neighboring
neurons (Kirchhoff et al. 2001, Fields e StevensGraham, 2002, Pekny e Nilsson, 2005). These
cells are suspect of being involved in a wide range of CNS
diseases, including trauma, ischemia, and neurodegeneration
(Muke e Eddleston, 1993). A well-known feature of
reactive astrocytes is increased production of intermediary
filaments (IFs) and the increased positivity of glial fibrillary acidic protein (GFAP), vimentin and also nestin, two
intermediate proteins that are only abundantly expressed
in immature astrocytes (Eng et al., 2000). According to the literature, the morphological
changes in glial cells (hypertrophy/hyperplasia) reflect the
increase in their functional capacity (Pekny e Nilsson,
2005). Although the characterization of astrocytic response
to brain injuries, neurodegenerative disease and in cell
transplantation are common subject of research, we still
know little about how astrocytes influence the regrowth or
reconstruction of neural network after the developmental
period. The overall impact of astrocytic presence on the
growth of neurons in the adult CNS needs to be established
(Gates e Dunnett, 2001).
One of the useful astrocyte markers is the
intracellular glicoprotein S100-β that belongs to the
calmodulin family of calcium-binding proteins. S100-β
is produced by astrocytes and acts as a trophic factor for
serotonin neurons (Whitaker-Azmitia et al., 1990,
Van Eldik e Wainwrigth, 2003) and also regulates
the assembly of intermediate filaments by inhibiting GFAP
polymerization in the presence of Ca+2 (Bianchi et al.,
1994, Kligman e Marshak, 1985, Mrak et al.,
1995). It has been suggested that S100-β has important
autocrine and paracrine action and is considered an
intercellular signaling. S100-β may be implicated in the
pathophysiology of brain disorders, through increased
release by astrocytes consequent to their activation and/
or death (Adami et al., 2001, Rothermundt et al.,
2003, Donato et al., 2003, Marenholtz, et al.,
2004). S100B stimulates iNOS in rat primary cortical
astrocytes through a signal transduction pathway that
involves activation of the transcription factor NFκB. The
NFκB family of proteins is one of the best characterized
transcription factors and regulates expression of many
genes involved in peripheral immune and inflammatory
responses. In recent years, an increasing body of evidence
also supports an inflammatory role for NFκB in the brain
(LAM et al, 2001).
Vesicular stomatitis virus (VSV) is a single
stranded, negative sense RNA virus, encased in a
bullet-shape virion made from only five proteins. Mice
experimental infection by the intranasal route results
in dissemination by retrograde transport in neurons
and ventricular surfaces (Hunneycutt et al., 1994,
Machado et al., 2003). Disease is dependent on dose,
route, and virus and has been used as a model for in vivo
and in vitro studies of acute encephalitis (Huneycutt et
al., 1994, Chen et al., 2001, Chen et al, 2002, Chesler
et al., 2004).
Some studies show rapid response of astrocytes
and microglia during acute VSV infection (Bi et al.,
109
1995, Vasconcelos et al., 2004). The relevance of
astrogliosis remains controversial, especially with respect
to the beneficial or detrimental influence of reactive
astrocytes on CNS recovery from inflammation studies
(Silver e Miller, 2004). Many studies were able
to demonstrate a correlation of traumatic brain injury,
hypoxic and ischemic brain damage and upregulation of
S100β and few data exists for inflammatory and infectious
diseases (Rothermundt et al., 2003). More knowledge
about the correlation of astrocyte activation and S100β
expression could help to understand the physiopathology
of acute encephalitis and recovery stage using the VSV
model. Reports on the VSV infection pathogenesis have
been pointed out in acute phase and there are many gaps
involving, for instance, aspects about recovery of infected
animals and the participation of glial cells in subacute and
in late stages.
The aim of the present study was to investigate
the immunoreactivity pattern of astrocytic markers, GFAP
and vimentin, correlating and comparing their levels with
S-100β localization during acute and in late phase following
intranasal inoculation of VSV in mice.
Material and Methods
Virus
The sample of vesicular stomatitis virus, strain
Indiana type II was maintained at -80ºC in BHK. Mouse
brain suspension used for inoculation was obtained from
intracerebral inoculation of mice as previous described
by Machado et al. (2003). The VSV titration determined
as infecting dose per tissue culture (TCID50/0.1 ml), was
105 virus/0.1ml.
Experimental infection of mice
Forty male C57BI6 mice, 5 to 7 weeks old
(CEMIB-UNICAMP-SP-Brazil), were used for viral
inoculation. Mice were mildly anesthetized in a closed
container with ether followed by intranasal inoculation
with VSV suspension (105 virus/0.1ml) in a total volume
of 0.03ml administered equally between each nostril according to Huneycutt et al. (1993). Four mice got
sterile PBS into the nostril and were used as control.
Animals were housed with food and water ad libitum and
were randomly sacrificed for histological and immunohistochemical analysis (6-12 mice/sacrifice point, Table1). To
minimize suffering to the animals they were sacrificed at
start of the paralysis symptoms.
Tree groups of inoculated mice were formed:
mice sacrificed on day 2 (Group 1), 6 (Group 2) and
20 (Group 3) post inoculation. Non-inoculated mice
composed group 4. All the protocols were approved and
experiments were carried out in agreement with UNESPAnimal Experimentation Ethics Committee. The animals
110
were housed with food and water at libitum.
Histological and Immunohistochemical analysis
Mice were anesthetized with ether and sacrificed
with intraperitoneal lethal dose of 5mg of pentobarbital
sodium (Nembutal® - Abbott Laboratories) in normal saline solution. Each mouse was perfused transcardially with
30-40ml of phosphate-buffered saline (PBS pH 7.4). After
perfusion whole brains were removed and fixed during 8
hours in freshly prepared 4% buffered paraformaldehyde
(pH 7.4) and embedded in paraffin according to standard
procedures. Five-micrometer brain sagittal sections (1-1.5
mm lateral from bregma) were stained with hematoxylin
and eosin in order to detect inflammatory, degenerative or
reactive features of neurons. For immunohistochemistry,
first endogenous peroxidase activity was blocked by incubating sections in 1% H2O2. Sections were treated with
Tripsin 1% at 37oC before blocking of nonspecific binding
with powdered skimmed milk (3% in phosphate-buffered
saline) for 30 minutes. Sections were then incubated
overnight at 4ºC with the anti-GFAP (SIGMA G9269,
1:300), anti-vimentin (Novocastra NCL-VIM-V9, 1:200)
and anti-S100β (Novocastra RTU-S100p). Biotinylated
antibodies anti-rabbit or anti-mouse (Dako, 1:100) were
applied as secondary antibodies and an avidin-biotinperoxidase complex (ABC kit, Vector Laboratories, USA)
served as the third reagent. DAB (3.3-diaminobenzidinetetrahydrocloride)-H2O2 served as chromogen. Sections
were counterstained with Mayer hematoxylin. Suppression
of primary antibodies incubation was used as control. Because most severe lesions are observed in the olfactory bulb
we choose this areas for study, together with frontal cortex,
subependimal plate and ependymal layer cells, diencephalon nucleus and neurons at brain stem (HUNEYCUTT
et al., 1994, Machado et al., 2003). Two independent
observers scored the immunohistochemical evaluation. The
extent of immunoreactivity for each of antibody used was
determined using an arbitrary scale of + to ++++ reflecting
the extent and the intensity of immunopositivity.
Results
At 2 days post inoculation mild meningitis can
be observed in the olfactory bulb area. Symptoms of encephalitis are visible on the 5th day post inoculation. Typical clinical signs observed on 6th day included ruffled fur,
conjunctivitis, less activity and decreased consciousness
and progressive posterior paralysis. Encephalic lesions
at this time included: leptomeningitis, which intensity
varied from mild to intense, mostly observed at olfactory
bulb and extended to encephalic ventral surface. Nearly
all affected mice showed neuronal death in the olfactory
bulb and focal malacea. Ventriculitis at laterals, third and
fourth ventricles affecting subependymal layer also was
remarkable feature. Perivascular infiltrates and neuronal
necrosis were also observed in thalamus, hypothalamus
and brain stem. Inflammatory infiltrate was composed of
neutrophils and lymphocytes. Mice without symptoms sacrificed on 6th day pi showed mild inflammation restricted
to olfactory bulb area. Animals that survive VSV infection
do not show any symptom or morphological alteration on
20 days pi.
Fibrous and protoplasmatic astrocytes positive
for GFAP were easily identified in animals of VSV inoculated (groups 1, 2, 3) or control (group 4). On the 2nd
day p.i., reactive astrocytes were observed in group 1 with
moderate staining particularly in white matter areas. The
astrocytes were characterized mainly for having large and
clear nucleus, clumped chromatin located peripherally,
and large nucleoli. The cytoplasm was evident and the
thick processes were intensively stained. Cells with those
characteristics were observed mainly in the olfactory bulb
(Figure 1 A), externally-limiting glia and in the glomerular and mitral cell layers. Six days after VSV inoculation
(group 2) we noticed diffuse, strong to intense GFAP
immunoreactivity (Table 1), with exception of reduction
in the GFAP intensity in some areas of the encephalon
that correspond to necrotic regions in mice sacrificed with
neurological symptoms. GFAP-positive cells, with large
swollen nuclei, evident nucleoli, and processes with a
fragmented aspect were observed in areas adjacent to necrotic areas, followed by transition areas where astrocytes
showed less marked degenerative morphological aspects,
i.e., the processes were short, thick and showed only an
initial fragmentation or dissolution (Figure 1B). In mice
sacrificed on day 6 without symptoms, the tissue injury
was less intense and mainly limited to the olfactory bulb,
where GFAP-positive astrocytes with activated morphology were observed. After 20 days (group 3), astrocytes
showed typical morphologic pattern and GFAP staining
was the same observed in the control group (group 4).
Vimentin expression in astrocytes did not appear in animals of control group except at Bergmann glia
and in spare cells in cortex. On day 2 post viral inoculation,
no difference in cell staining was observed. Six days post
inoculation, strong vimentin immunostaining was detected in round cells with macrophagic appearance in the
olfactory bulb (Figure 1C) and in the cytoplasm and thick
processes of astrocytes throughout cortex and subpial glia
of frontal cortex (Figure 1D). Only few astrocytic cells in
the hippocampus, thalamus and brain stem were vimentin
positive and Bergmann glia showed a reduction in vimentin
stain intensity. Inflammatory cells in the leptomeninges
and cerebral ventricles were also vimentin positive. Twenty
days after inoculation vimentin, labeling returned to the
pattern observed in non-inoculated mice (group 4) and in
mice from group 1 (2 days post inoculation).
S100β immunohistochemical staining was faint,
diffuse and localized in the neuropil in groups 1, 3 and
4 (control). In these groups just a few cortical neurons
showed S100-β cytoplasmatic positivity. S100β protein
staining was evidently detected only six days after inoculation (Group 2) and especially in the brain of mice that
showed neurological symptoms. The stain was observed
in the cytoplasm of neurons in the olfactory bulb (Figure
1E), in microglial cells (Figure 1F), in subependymal
plate and ependymal layer cells, in neurons in the frontal
cortex, diencephalon nucleus and brain stem. In these
brains, the intensity was scored as strong staining and the
pattern of distribution and intensity is showed in Table
1. Mice without symptoms on day 6 showed diffuse pattern of positivity in neuropil of the olfactory bulb and
periventricular areas.
Discussion
Previous studies showed that two days after
inoculation VSV antigen was detected in the olfactory
bulb of mice, and on day six post inoculation, the viral
antigen was disseminated in areas such as periventricular
ependyma, hippocampus, thalamus, hypothalamus, mesencephalon nuclei, and brain stem nuclei (Huneycutt
et al. 1994, Machado et al., 2003, MACHADO et al.,
2006). These areas showed correspondence with those
where we observed, on six days post inoculation, neuronal
degeneration and more intense astrocytic reaction characterized by intense GFAP reactivity. The semi-quantitative
results of GFAP, vimentin and S100β detection are showed
on Table 1. Astrocytic response is commonly seen where
tissue damage has occurred (Pekny e Pekna, 2004,
Panickar e Noremberg, 2005). We noticed decrease
in GFAP detection in astrocytes together with fragmentation
on their processes in surrounding areas of necrotic tissues.
At the same areas, vimentin staining was stronger and most
evident than GFAP. In areas where initially viral replication
takes place (olfactory bulb) we detected positive cell, to both
GFAP and vimentin. Vimentin is a mesenchimal intermediate filament that characterizes immature astrocytes (Bignami et al., 1982). After birth, vimentin is progressively
replaced by GFAP as a key step in the differentiation of
astrocytes (Bovolenta et al., 1984). The co-expression
of vimentin and GFAP is, however, retained is some astroglial population in normal adulthood (Lazarides, 1982)
and is upregulated in response to injury in the CNS. This
explains why the less positive vimentin stain was observed
in animals after 2 and 20 days of VSV inoculation (groups
1 and 3) and also in normal mice (group 4) and the strong
detection observed during the acute inflammatory phase.
In addition to astrocytes, round cells with macrophage morphology were vimentin positive. Yamada et
al. (1992) described similar microglial behavior in pathological human brain. Round microglia stained with OX-42
111
antibody was described by Vasconcelos et al. (2004),
in necrotic areas during VSV encephalitis, confirming the
presence of those cells in VSV induced lesion.
Because astrocytes function as a syncytium of
interconnected cells both in health and in disease, rather
than as individual cells, any disturb at a specific region could
reflect in the whole astrocyte population (Pekny e Nilsson, 2005). Both fragmentation and reducing of GFAP
observed in astrocytes during VSV encephalon infection
may also represent a signal for structural deficit of mature
astrocytes, including blood brain barrier breakdown, as observed by Bi et al. (1995). These morphological changes also
represent disruption of astrocyte modulating functions and
probably contribute to the worsening of the lesions caused
by inflammation in agreement with Muke e Eddleston
(1993) observations.
The S100β protein was strongly stained in the
brains of mice mostly during the acute phase of VSV encephalitis. Although it has been described as an exclusive
marker for astrocytes, S100-β has also been localized
in neurons and other cells of different areas in the brain
of some mammals including humans (Rickmann e
Wolff, 1995, STEINER et al., 2007). In infected symptomatic mice, we observed neuronal cells with positive
stain against S100-β in areas that match with those where
viral dissemination that was previous described (HUNEYCUTT et al., 1994, Machado et al., 2003, MACHADO
et al., 2006) and where astrocytes are activated, contiguous
to necrotic areas. As reported in advance, depending on
the disease context and on the concentration, S100-β can
be viewed as beneficial to promote neuronal survival or it
can be seen as detrimental for neuronal function. Serum
protein S100-β determination is considered a sensitive
marker of brain injury (Savola et al., 2004), and reflects
the degree of blood-brain barrier opening (Kleindienst
et al., 2004). A beneficial effect of S100-β intraventricu-
lar infusion was observed on recovery of rats with brain
traumatic injury (Kleindienst et al., 2004) and in a
biaxial strain model in vitro injury (ELLIS et al., 2007).
At the low levels normally found in the brain extracelullar
space, S100-β acts as a neurotrophic factor protecting
neurons against noxious stimuli and stimulating neurite
outgrowth (Ueda et al., 1995, Barguer et al., 1995,
Iwasaki et al., 1997). We detected faint extracellular
staining in encephalons of mice in groups 1, 3 and 4 suggesting a constitutive basal production in mice brain. Low
concentrations of extracelullar S100-β induce astrocytic
proliferation in vitro (Selienfreund et al., 1991).
However, elevated concentrations (i.e. micromolecular)
of S100-β induce the expression of iNOS in astrocytes
(Hu et al., 1996), that can directly induce neuronal death
via apoptosis (Kronke et al., 1997). As the most severe
neuronal lesions are detected in the olfactory bulb of
symptomatic mice at day 6p.i., we believe that the strong
neuronal detection of S100β at this day might be associated with induced neuronal death.
Although generally assumed as an astrocyte
marker S100β was not detected by immunohistochemistry
during acute phase of VSV encephalitis. The fail in detect
S100β staining in astrocytes could be related with the cell
compromise of production of another cytokine as TGFβ1
(MACHADO et al., 2006) or as have been suggested
by Ohtaki et al (2007) the viral infection may impair
astrocyte function via a downregulation of S100β and
interference with inflammatory response.
In conclusion, during VSV infection in the mouse
model and especially in those mice with symptoms at day
six post inoculation we showed that besides astrocytes
response to VSV infection characterized by upregulation
of GFAP and vimentin, there is also S100β production
detected in neurons and microglia, but not in astrocytes,
where S100β is considered a specific marker.
Table 1 - Summary of general immunohistochemical staining patterns of GFAP, vimentin and S100-β in brain of mice
inoculated and sacrificed on 2, 6 and 20 days post inoculation and normal control. Semi-quantitative results of
observations in the olfactory bulb, frontal cortex, subependymal plate and ependymal layer cells, diencephalon
nucleus and neurons in brain stem are showed.
Astrocytes markers
Days post VSV
inoculation
GFAP
Vimentin
S100-β
Symptoms
2 (n=12)
++
+
+
No (n=12)
++++
+++
+++
Yes (n=5)
6 (n=12)
+++
++
++
No (n=7)
20 (n=12)
+
+
+
No (n=12)
Control (n=4)
+
+
+
No (n=4)
faint staining, ++ moderate staining, +++ strong staining, ++++intense staining
112
Barguer, S. W., Van Eldik, L. W., Mattson, M.
P. S100β protects hippocampal neurons from damage
induced by glucose deprivation. Brain Research, v.677,
p.167-170, 1995.
Bi, Z., Barna, M., Komatsu, T., Reiss, C. S. Vesicular stomatitis virus infection of the central nervous system
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Bianchi, R., Verzini, M., Garbuglia, I, GiaMbanco, I, Donato, R. Mechanism of S100 protein
dependent inhibition of glial fibrillary acidic protein
(GFAP) polymerization. Biochemistry and Biophysical
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Bignami, A., Raju, T, Dahl, D. Localization of vimentin, the nonspecific intermediate filament protein, in
embryonal glia and in early differentiating neurons. In vivo
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with vimentin and neurofilament antisera. Developmental
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Figure 1: A - Olfactory bulb of mice from control group
showing GFAP positive astrocytes with normal morphology. Note small nuclei and thin processes and compare
with astrocytes in B. From B to F - GFAP, vimentin and
S100-β immunostaining in mouse encephalon observed at
6 days post VSV inoculation. B - Astrocytes in the olfactory bulb has vesicular nucleus (arrow) and fragmented
processes surrounding necrotic area where GFAP staining
intensity is faint. C – Phagocyte round cells within olfactory bulb with intense staining for vimentin (arrow) D –
Strong vimentin immunoreactivity in astrocytes at frontal
cortex. E – Olfactory neurons showing S100-β positive
stain (arrows). F – Microglial cells in frontal cortex with
positive staining for S100β (arrow). Sagittal section, ABC
method, A-F: Bar =12.5 µm
Acknowledgements
We are grateful to FAPESP and FUNDUNESP
for financial support.
ARTIGO RECEBIDO: Março/2006
APROVADO: Outubro/2007
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